Method of diminishing the adverse effects of interleukin-2

ABSTRACT

A method and composition for preserving or enhancing the beneficial effects of interlude-2 (IL-2) while simultaneously mitigating the adverse effects of IL-2 on a subject is disclosed. The method involves administering an amount of a leukotriene B 4  (LTB 4 ) antagonist, preferably a LTB 4  receptor antagonistic, to a subject exhibiting adverse pharmacological effects due to exogenous IL-2, where the amount administered to the subject is sufficient to decrease the IL-2 induces adverse effects. Also disclosed is an article of manufacturing comprising a composition of the leukotriene B 4  antagonist in combination with labeling instructions for treatment. Also disclosed is a method for preparing a pharmaceutical composition. Generally the LTB 4  antagonist will reduce the IL-2-induced adverse effects while preserving or enhancing the anti tumor, antiviral, or immunostimulatory effects of IL-2, thereby improving the therapeutic index of IL-2.

CROSS REFERENCE

[0001] This is a continuation-in-part of U.S. Ser. No. 09/334,276, filedJun. 16, 1999, which in turn claims priority to and is acontinuation-in-part of U.S. Ser. No. 60/098,341, filed Aug. 28, 1998.Both applications are incorporated herein by reference.

INTRODUCTION

[0002] 1. Technical Field

[0003] This invention relates to mitigating adverse effects induced byinterlude-2 through the administration of an effective amount of aleukotriene B₄ antagonist.

Background

[0004] Recombinant interleukin-2 (PROLEUKIN® or “IL-2”) is an analogueof human native interleukin-2. While human native interleukin-2 ispresent in a human in small amounts, under certain conditions, i.e., theadministration of IL-2 to treat certain conditions, excess levels (i.e.,higher than normal levels) of IL-2 will be present in a subject'ssystem. IL-2 is approved for the treatment of certain human malignanciesincluding melanoma and renal cell carcinoma and is useful in treatingcertain viral conditions. IL-2 is thought to work by stimulating theimmune system to work as an antitumor or antiviral agent. It can beviewed as having antitumor, antiviral, and immunostimulating activity.The administration of IL-2 has been associated with “vascular leaksyndrome” (VLS), which results from extravasation of plasma proteins andfluid into the extravascular space. It is known that, among otheradverse signs or symptoms, VLS can cause generalized edema, systemichypotension, reduced organ perfusion, and subsequent dysfunction of oneor more organs. When sufficiently severe, VLS may cause significantdisability or even death. The adverse effects of IL-2 may necessitateusing a lower dose of IL-2, thereby diminishing the potential fortherapeutic benefit from IL-2. An effective means of mitigating IL-2toxicity would be beneficial.

[0005] Empirical approaches to treating VLS have included the use ofcorticosteroids, which, unfortunately, can reduce the antitumor effectsof IL-2. The pluripotent filaricide diethylcarbamazine (DEC) has alsobeen studied experimentally, but because of its diverse pharmacologicaleffects, the specific role of DEC in VLS is unclear. DEC is known to bea 5-lipoxygenase inhibitor. The antifolate methotrexate has also beensuggested as a means of mitigating VLS. Unfortunately, methotrexateitself is quite toxic.

[0006] However, blocking the adverse effects of any substance, e.g.IL-2, may also block the beneficial effects. It is a deficiency of priorart that no intervention to prevent or mitigate VLS has been shown notto interfere with the antitumor activity or, more preferably, to enhancethe antitumor activity of interleukin-2.

[0007] It is generally known that 5-lipoxygenase is instrumental ininitiating production of 5-hydroperoxyeicosatetraenoic acid (HPETE) andleading to the production of 5-HETE, LTA₄, LTB₄, LTC₄, LTD₄, and LTE₄.See FIG. 3-18 at page 71 of Robbins, Pathologic Basis of Disease, 6thEd. By Ramzi S. Kotran, M.D.; Vinay Kumar, M.D., FRC Path.; and TuckerCollins, M.D., Ph.D., W. B. Saunders & Co. (1999). This Chapter alsoestablishes that LTC₄, LTD4, and LTE₄ have an action of elevatingvascular permeability, but not LTB₄. (See Table 3-4). See Goodman &Gilman's, The Pharmacological Basis of Therapeutics, 9th Ed., McGrawHill (1996), pp. 601-616. On cell activation and intracellularCa²⁺increases, 5-lipoxygenase binds to a 5-lipoxygenase activatingprotein (FLAP). This binding activates the 5-lipoxygenase which resultsin its association with the cell membrane and increased synthesis of5-HPETE. The 5-HPETE is rearranged by LTA synthase to an unstable5,6-epoxide, known as LTA₄. LTA₄ may be transformed by LTA hydrolase toLTB₄. Alternatively, it may be conjugated with glutathione by LTC₄synthase to form LTC₄. LTC₄ may be transformed to LTD₄ by the removal ofglutamic acid. LTD₄ may in turn be converted to LTE₄ by the cleavage ofglycine. The reincorporation of glutamic acid to LTE₄ yields aγ-glutamylcysteinyl derivative called LTF₄.

[0008] Thus, a 5-lipoxygenase inhibitor would be expected to decreaseproduction of 5-HPETE (and 5-HETE), LTA₄, LTB₄, LTC₄, LTD₄, and LTE₄.

[0009] As the variety of potential interventions suggests, thepathogenesis of VLS is unknown. It has been demonstrated, however, thatIL-2 increases plasma levels of leukotriene B₄. I have found that usinga leukotriene B₄ receptor antagonist (LA), preferably a leukotriene B₄antagonist, during IL-2 therapy mitigates VLS and lessens the adverseeffects of IL-2 while preserving or enhancing the beneficial effect ofIL-2.

[0010] Thus, evidence shows that specific blockade of the leukotriene B₄receptor not only mitigates VLS and lessens the adverse effects of IL-2but it also preserves or enhances antitumor, antiviral, orimmunostimulatory activity, thereby improving the therapeutic index ofIL-2. The improved therapeutic index of IL-2 leads to severaladvantages. These include

[0011] reducing diagnostic testing required to

[0012] i. enhance tumor response to IL-2 by permitting a higher numberof doses or a larger dosing amount of IL-2 with no significant increasein adverse effects induced by IL-2

[0013] ii. monitor patient responses to IL-2

[0014] iii. determine the success of therapeutic interventions requiredto mitigate IL-2 related adverse events

[0015] iv. demonstrate that certain events are caused by IL-2 ratherthan another agent

[0016] obviating or reducing the need to place patients into intensivecare units or onto respirators in the case of severe pulmonary edema,

[0017] obviating or reducing the need to place patients into cardiac orcoronary care units in the case of severe arrhythmia or congestive heartfailure,

[0018] obviating the need for placing patients onto dialysis protocolsthe case of renal compromise,

[0019] reducing the need for intensive nursing or support care.

[0020] reducing or obviating the use of medications or other devices forprevention or treatment of adverse effects expected or caused by IL-2

[0021] All of these advantages should then lead to better toleratedtreatment, reduced overall cost of treatment and better response totreatment.

SUMMARY OF THE INVENTION

[0022] One aspect of this invention is a method of mitigating an adversepharmacological effect of IL-2 in a subject. The method comprisesadministering to a subject receiving exogenous IL-2 an amount of aleukotriene B₄ inhibitor or antagonist (preferably a leukotriene B₄receptor antagonist) that is sufficient to mitigate the adverse effects.Another aspect is treating a malignancy or viral infection in a mammalby administering a therapeutically effective amount of IL-2 incombination with a leukotriene B₄ antagonist in an amount sufficient toreduce IL-2 -induced adverse pharmacological effects, e.g., increasedvascular permeability. Another aspect is simultaneously preserving orenhancing the antitumor, antiviral, or immunostimulatory activity ofIL-2.

[0023] The preferred leukotriene B₄ receptor antagonist is representedby the Formula (I), or a stereoisomer or pharmaceutically acceptablesalt thereof, wherein

[0024] R¹ represents alkyl having 2 to 6 carbon atoms, alkenyl having 2to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, or (CH₂)_(n)Rwherein R represents cycloalkyl of 3 to 5 carbon atoms and n is 1 or 2;

[0025] R² represents hydrogen, methyl or ethyl;

[0026] R³ represents alkyl having 1 to 5 carbon atoms;

[0027] W represents a bridging group such as (CH₂)_(x) where x is 2 to7, alkenylene having 3 to 7 carbon atoms, alkynylene having 3 to 7carbon atoms or cyclopentyl;

[0028] R⁴ represents hydrogen, alkyl having 2 to 5 carbon atoms, alkynylhaving 2 to 5 carbon atoms, alkenyl having 2 to 5 carbon atoms, alkanoylof 2 to 5 carbon atoms, or aralkanoyl of 7 to 9 carbon atoms;

[0029] R⁵ represents hydrogen, alkyl having 1 to 6 carbon atoms, or R⁵represents alkanoyl having 2 to 4 carbon atoms, or (CH₂)_(y)-CO₂R⁸wherein y is 0 to 4 and R⁸ is hydrogen or alkyl having 1 to 6 carbonatoms;

[0030] R⁶ represents hydrogen or together with R⁵ represents a carbon tocarbon bond; and

[0031] A represents -Z-CO₂R⁷ wherein R⁷ represents hydrogen or alkylhaving 1 to 6 carbon atoms, and wherein Z is absent or representsstraight or branched chain alkylene or alkenylene having up to 6 carbonatoms.

[0032] Other leukotriene B₄ receptor antagonists include those set forthin U.S. Pat. No. 4,788,214 (which is incorporated herein by reference),BIIL-284 and ebselen (DR-3305) as further described herein.

[0033] Another aspect of the invention may be viewed as an improvementin a method of treatment. In a method of treating a malignancy or viralinfection or immunological disorder in a mammal, which method comprisesadministering a therapeutically effective amount of IL-2, whichadministration also induces adverse pharmacological effects in themammal, the improvement comprises administering a leukotriene B₄antagonist in an amount sufficient to mitigate IL-2 induced adversepharmacological effects, preferably while simultaneously preserving orenhancing the antitumor, antiviral, or immunostimulatory activity, ofIL-2.

[0034] Another aspect of this invention is a process for preparing apharmaceutical composition. The process comprises combining aleukotriene B₄ antagonist with a pharmaceutical excipient to form acomposition useful for mitigating IL-2 induced, adverse pharmacologicaleffects in a subject receiving exogenous IL-2, preferably whilesimultaneously preserving or enhancing the antitumor or antiviralactivity, or other desirable pharmacological activity, of IL-2.

[0035] Still another aspect of this invention is an article ofmanufacture that comprises a pharmaceutical composition comprising aleukotriene B₄ antagonist as a unit dosage in combination withinstructions for administering the composition to a mammal undergoingtreatment of a malignancy or viral infection with IL-2, wherein theamount of the composition administered is sufficient to mitigate IL-2induced adverse pharmacological effects in the mammal being treated,preferably while simultaneously preserving or enhancing the antitumor orantiviral activity, or other desirable pharmacological activity, ofIL-2. Still another aspect of this invention is an article ofmanufacture that comprises packaging and labeling thereof such that aleukotriene B₄ antagonist, preferably a leukotriene B₄ receptorantagonist, is used in conjunction with IL-2 for the treatment of IL-2responsive disorders.

[0036] In general, the LTB₄ antagonist, preferably the LTB₄ receptorantagonist, will reduce the adverse effects caused by IL-2 whilesimultaneously preserving or enhancing the antitumor, antiviral, and/orimmunostimulatory effects of IL-2 or will reduce the IL-2-inducedadverse effects more than any reduction in IL-2 related antitumor,antiviral, or immunostimulatory effects IL-2 . The use of the LTB₄antagonist can be said to improve the therapeutic index of IL-2.

DESCRIPTION OF THE FIGURES

[0037]FIG. 1: This figure presents comparative in vivo results showingthe effects of a compound useful in this invention on the reduction ofoxygenation of arterial blood by IL-2 administration.

[0038]FIG. 2: This figure presents the number of doses of IL-2 thatcould be safely administered to patients with metastatic renal cellcancer as related to the plasma level of a leukotriene B₄ antagonistuseful in this invention. As leukotriene B₄ antagonist plasma levelrises, the number of well-tolerated doses of IL-2 also increases(p<0.03).

DETAILED DESCRIPTION AND PRESENTLY PREFERRED EMBODIMENTS

[0039] For purposes of this application the following definitions apply:

[0040] Alkyl means a fully saturated hydrocarbon radical having thenumber of carbon atoms indicated. For example, alkyl of 1 to 6 includes,e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl,n-pentyl, amyl, n-hexyl, and the like.

[0041] Alkenyl means a radical derived from an alkene, i.e., hydrocarbonhaving a single double bond and having the number of carbons indicated.For example, alkenyl of 2-6 carbon atoms includes radicals derived fromethylene, propylene, 1-butene, 2-butene, isobutylene, 3,3-dimethylpropylene, and the like.

[0042] Alkynyl means a radical derived from an alkyne, i.e., hydrocarbonhaving a single triple bond and having the number of carbons indicated.For example alkynyl of 1-6 carbon atoms includes acetylenyl, propynyl, 1-butynyl, 1 pentynyl, 1-hexynyl and the like.

[0043] Alkanoyl means a radical represented by the formula RC(O)-where Ris alkyl of the number of carbons indicated.

[0044] Aralkanoyl means a radical represented by the formulaArC(O)-where Ar is an aryl group of the number of carbons indicated,e.g., phenyl (6 carbons).

[0045] LA is the abbreviation for leukotriene B₄ antagonist.

[0046] Pharmaceutically-acceptable salts are those that arephysiologically acceptable for pharmaceutical purposes and include,e.g., ammonium, potassium, sodium, alkaline earth, and the like.

[0047] A stereoisomer is one of a set of isomers whose molecules havethe same atoms bonded to each other but differ in the way these atomsare arranged in space. Included in this are enantiomers, i.e., compoundsthat are mirror images of each other but that are not superimposableupon each other.

[0048] It should be understood that the use of the alternative “or” withitems in a series is meant to include both the alternative and thecollective. Thus, “preserving the antitumor, antiviral, orimmunostimilatory effects” would include preserving each alone or in anycombination.

Compounds Useful in the Invention

[0049] An LA useful in this invention is one that blocks the effectsmediated by the leukotriene B₄ receptor. An LA may block the effectsmediated by the LTB₄ receptor by acting directly on the receptor or byinhibiting the synthesis of LTB₄, preferably the former. Non-limitingexamples of LTB₄ antagonists include in BIIL-284 and ebselen. BIIL-284is a drug being developed by Boehringer Engelheim in Germany. Thestructure of the compound is shown in formula (II) as follows:

[0050] Ebselen is being developed by Daiichi Pharmaceutical in Japan andis also known as DR-3305 and Harmokisane. It is an orally activeorganoselenium compound having the formula (III)

[0051] More information about these compounds may be found atPharmaprojects, accession numbers 17685 and 5076.

[0052] A preferred class of compounds is represented by Formula (I) asset forth in the “Summary of the Invention” section of this applicationor a pharmaceutically acceptable salt thereof. In Formula (I),

[0053] represents hydrogen, alkyl having 1 to 6 carbon atoms, alkenylhaving 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, or(CH₂)_(n)R wherein R represents cycloalkyl of 3 to 5 carbon atoms and nis 1 or 2;

[0054] R² represents hydrogen, methyl or ethyl;

[0055] R³ represents alkyl having I to 5 carbon atoms;

[0056] W represents (CH₂)_(x) where x is 2 to 7, alkenylene having 3 to7 carbon atoms, alkynylene having 3 to 7 carbon atoms or cyclopentylene;

[0057] R⁴ represents hydrogen, alkyl having 2 to 5 carbon atoms, alkynylhaving 2 to 5 carbon atoms, alkenyl having 2 to 5 carbon atoms, alkanoylof 2 to 5 carbon atoms or aralkanoyl;

[0058] R⁵ represents hydrogen, alkyl having 1 to 6 carbon atoms, or R⁵represents alkanoyl having 2 to 4 carbon atoms, or (CH₂)_(y)-CO₂R⁸wherein y is 0 to 4 and R⁸ is hydrogen or alkyl having 1 to 6 carbonatoms;

[0059] R⁶ represents hydrogen or together with R⁵ represents a carbon tocarbon bond; and

[0060] A represents-Z-CO₂R⁷ wherein R⁷ represents hydrogen or alkylhaving 1 to 6 carbon atoms, and wherein Z is absent or representsstraight or branched chain alkylene or alkenylene having up to 6 carbonatoms.

[0061] A preferred subgroup is represented by Formula (I) wherein R¹represents alkyl having 2-4 carbon atoms; R² represents hydrogen, methylor ethyl; R³ represents alkyl having 1 to 3 carbon atoms; W represents(CH₂)_(x) where x is 3 to 5, alkenylene having 3 to 5 carbon atoms,alkynylene having 3 to 5 carbon atoms, or cyclopentylene; R⁴ representsalkyl having 2 to 4 carbon atoms, acetyl or benzoyl; R⁵ representshydrogen, or alkyl having 1 to 4 carbon atoms; and A represents-Z-CO₂R⁷,wherein R⁷ represents hydrogen or alkyl having 1 to 4 carbon atoms, andwherein Z is absent or represents alkylene having up to 2 carbon atoms;or a stereoisomer or pharmaceutically acceptable salt thereof of thissubgroup. Particularly useful are compounds represented by Formula (I)wherein R¹ is n-propyl; R² and R³ each is methyl; W is (CH₂)_(x), wherex is 3,4 or 5; R⁴ is n-propyl; R⁵ represents hydrogen or alkyl of 1 to 4carbon atoms; and A represents -Z-CO₂R⁷ wherein R⁷ represents hydrogenor alkyl having 1 to 4 carbon atoms and Z is absent or representsalkylene having up to 2 carbon atoms; or a stereoisomer or apharmaceutically acceptable salt thereof. Particularly preferred is aleukotriene B₄ receptor antagonist as represented by Formula (I) whereinR¹ is n-propyl, R² and R³ each is methyl, R⁴ is n-propyl at the8-position. W is (CH₂)₃; R⁵ is H; and A is (CH₂)_(p)-COOH, where p is0,1 or 2, especially 0 or a stereoisomer or a pharmaceuticallyacceptable salt thereof.

[0062] Other compounds useful in this invention are set forth in U.S.Pat. No. 4,788,214.

[0063] The compounds represented by Formula (I) in which position 2 ofthe benzopyran ring is a chiral center exist in racemic form as amixture of individual enantiomers (i.e., racemate), or as the pureindividual enantiomers. Formula (I) is intended to cover the racemicmixture containing equal quantities of dextrorotatory (+) andlevorotatory (−) enantiomers as well as the individual dextroratoryenantiomer, the levorotatory enantiomer and other non-equal mixtures ofenantiomers. The formula is to be interpreted as covering anystereoisomer of the compound.

[0064] Representative compounds include the following (or thecorresponding pharmaceutically acceptable salts thereof or thestereoisomers):

[0065] 7-[3 -(4-acetyl-3 -methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid;

[0066] 7-[[5-(4-acetyl-3-methoxy-2-propylphenoxy)pentyl]oxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid,ethyl ester;

[0067] 7-[3 -(4-acetyl-3 -methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8 -propyl-2H-1-benzopyran-2-propanoic acid;

[0068] 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-2-methyl-8-propyl-2H-1-benzopyran-2-propanoic acid;

[0069] 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-2-methyl-8-propyl-2H-1-benzopyran-2-propanoic acid,methyl ester;

[0070] 7-[3-(4-acetyl-2-(cyclopropylmethyl)-3-methoxyphenoxy)propoxy]-3,4dihydro-8-propyl-2H- 1 -benzopyran-2-carboxylic acid;

[0071] 7-[[5-(4-acetyl-3-methoxy-2-propylphenoxy)pentyl]oxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid;

[0072] 7-[5-(4-acetyl-3-hydroxy-2-propylphenoxy)-pentyloxy]-6-acetyl-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid; and

[0073] 7-[5-(4-acetyl-3-hydroxy-2-propylphenoxy)-pentyloxy]-6-benzoyl-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid.

[0074] Preparation of Compounds Useful in this Invention

[0075] These compounds, previously unknown for use with IL-2, areprepared by processes set forth in U.S. Pat. No. 4,889,871 issued Dec.26, 1989 to Djuric, et al. and U.S. Pat. No. 4,788,214 issued Nov. 29,1988 to Cohen et al. These patents are incorporated herein by referencein their entirety. The compounds useful in this invention are preparedby methods that result in the racemic compound or by stereospecificmethods that result in the individual stereoisomers.

[0076] U.S. Pat. No. 4,665,203 issued May 12, 1987 discloses methods formaking some of the intermediates used in making compounds of the presentinvention. The patent is incorporated herein by reference.

[0077] Preferred compounds useful in this invention, where R² is methylor ethyl, are generally prepared by alkylating the prior art phenolhydroxy (R² is H) compounds to form compounds of Formula (I) byconventional techniques. Thus, the reaction of the phenol hydroxy (R2 isH) with methyl iodide in potassium carbonate provides the ether.Dimethyl sulfate in acetone and base is also useful in preparing ethers.Alternatively, intermediates can be alkylated prior to forming the—O—(CH₂)_(x)—O—bridge. Hydrolysis of the ester compounds in the presenceof lithium hydroxide and methanol gives the acid compounds.

[0078] The compounds of Formula (I) and those of U.S. Pat. No. 4,788,214that contain an asymmetric carbon atom at position 2 of the benzopyranring are normally obtained from the synthesis as racemic mixtures.Resolution of the racemates into the corresponding optically activeisomers (enantiomers) can be carried out by persons skilled in the artusing known procedures.

[0079] Compounds of Formula (I) when R⁷ is hydrogen are carboxylicacids. A racemic mixture of a carboxylic acid may be resolved by firsttreating the racemate with an optically active amine base to form amixture of diastereomeric salts. Examples of optically active aminebases that may be used for this purpose are (R)-(+)-∝-methylbenzylamine,(S)-(−)-∝-methylbenzylamine, (1R,2S)-(−)-ephedrine, quinine, andquinidine. The thusly formed diastereomeric salts have differentproperties, such as solubility, and the diastereomers may therefore beseparated by selective recrystallization from a suitable solvent. Theoptically active carboxylic acids may then be obtained byre-acidification of the separated diastereomeric salts.

[0080] Alternatively, a racemic mixture of a carboxylic acid may betreated with an optically active alcohol to form a mixture ofdiastereomeric esters. Examples of optically active alcohols that may beused for this purpose are (1R,2S,5R)-(−)-menthol,(1S,2R,5S)-(+)-menthol, (R)-(−)-2-octanol, and (S)-(+)-2-octanol. Thethusly-formed mixture of diastereomeric esters may then be separated bychromatography. The optically active carboxylic acids may then beobtained from the separated diastereomeric esters by conventionaltechniques, such as treatment of the esters with sodium hydroxide orlithium hydroxide followed by reacidification.

[0081] Compounds of Formula (I) when R⁷ is alkyl are esters. A racemateof the esters may be resolved into the enantiomers by first resolving aracemic mixture of the corresponding carboxylic acid using one of themethods described above. The optically active ester may be obtained byesterification of the corresponding optically active carboxylic acid byprocedures similar to those used to prepare a racemic ester.

[0082] Alternatively, a racemic mixture of a carboxylic acid of Formula(I) or a racemic mixture of an ester of Formula (I), may be separatedinto the individual enantiomers by high performance liquidchromatography using a suitable chiral stationary phase and a suitableeluent.

[0083] Administration of Compounds Useful in this Invention

[0084] An aspect of this invention is a method of mitigating an adversepharmacological effect of IL-2 in a subject at risk of, or in factexhibiting, such effect. The method comprises administering to a subjectreceiving IL-2 an amount of an LA, preferably a leukotriene B₄ receptorantagonist, that is sufficient to mitigate the adverse effects of theIL-2, preferably while preserving or enhancing the benefits of IL-2.Generally the excess IL-2 is a result of IL-2 (e.g., PROLEUKIN®)administration to treat a malignancy, acquired immunodeficiency syndrome(AIDS) or other malady.

[0085] To mitigate the adverse effects induced by IL-2 administrationwhile preserving or enhancing benefits from IL-2 and thereby improvingthe therapeutic index of IL-2, a compound is delivered at a levelsufficient to achieve both goals simultaneously. That amount will varysomewhat from subject to subject but generally will be in the range ofabout 0.1 mg to about 10.0 mg per kilogram of body weight per day. Thepreferred range is from 0.1 to 5.0 mg/kg/day while the most preferredrange is from 1.0 to 5.0 mg/kg/day. Thus, for a 50 kg person, about 5.0to 500 mg/day would be administered. For a 70 kg person about 7.0 to 700mg/day.

[0086] In general, the adverse pharmacological effect of IL-2 in asubject will occur during or after the treatment of the subject for anIL-2-responsive disease state. Thus, the method, along with otheraspects of the invention, is useful in treating a subject having aleukotriene B4 receptor in its system. This generally includes mammals,such as livestock and pets, and particularly humans. Thus, thisinvention will find use in treating humans of all ages as well as intreating animals, i.e., in veterinary uses. The invention may be usedfor treating livestock such as cattle, sheep, pigs, goats, and the likeor for treating household pets such as dogs, cats, rabbits, hamsters,mice, rats, and the like. The primary utility is for treating humans.IL-2 is administered to a human as part of the treatment of a malignanttumor, i.e., cancer, or a viral disease such as AIDS. The adversepharmacological effect often seen in such treatment is increasedvascular permeability, e.g., VLS. The signs and symptoms of the adversepharmacological effect are, for example, cardiovascular (hypotensionrequiring pressors; arrhythmias, pericardial effusion); pulmonary(congestion, dyspnea, pulmonary edema, hypoxemia); hepatic (increasedbilirubin, jaundice, ascites); hematologic (anemia, thrombocytopenia,leukopenia); gastrointestinal (nausea, emesis, diarrhea,gastrointestinal bleeding); renal (oliguria/anuria, decreased excretoryfunction); dermatologic (pruritus, erythema, rash); musculoskeletal(arthralgia, myalgia); neural (dysfunction of central or peripheralnervous system, epileptic seizures); general (fever, pain, fatigue,weakness, localized or generalized edema, infection, weight gain,headache).

[0087] Thus, another aspect of this invention is a method of treating amalignancy or viral or immunological disease in a mammal, which methodcomprises administering a therapeutically effective amount of IL-2 inconjunction with the LA, as described herein, in an amount sufficient tomitigate IL-2-induced adverse physiological effects. The method may beperformed by administering the IL-2 and the LA in combination as a unitdosage or the IL-2 and the LA may be administered individually, with theLA being administered before, during or after the administration of theIL-2. The LA may be administered by any suitable route ofadministration, preferably parenterally orally before, during or afterthe IL-2 is administered.

[0088] Another aspect of this invention is a method of treating amalignancy or viral or immunological disease in a mammal and improvingthe therapeutic index of treatment, which method comprises administeringa therapeutically effective amount of IL-2 in conjunction with the LA,as described herein, in an amount sufficient to mitigate IL-2 inducedadverse effects while preserving or enhancing the antitumor, antiviral,or immunostimulatory activity of IL-2. Those skilled in the art willrecognize that this method will increase the therapeutic index oftreatment.

[0089] Another aspect of this invention comprises administering atherapeutically effective amount of IL-2 in conjunction with the LA, asdescribed herein, in an amount sufficient to reduce IL-2 adverse effectsmore than any reduction in antitumor, antiviral, or immunostimulatoryeffects, thereby also enhancing the therapeutic index of treatment.

[0090] Viewed another way, this invention may be seen as an improvementin a method of treating a malignancy or other malady requiring IL-2treatment. The subject method comprises administering a therapeuticallyeffective amount of IL-2. Because this treatment induces adversepharmacological effects in the mammal, the improvement of this inventioncomprises administering the LA in an amount sufficient to mitigate IL-2induced adverse pharmacological effects, preferably while preserving orenhancing the efficacy of IL-2.

[0091] Those skilled in the art will also recognize the currentinvention would also be useful when a chemically or biologicallymodified form of interleukin-2 is employed, wherein such modification initself is intended to reduce the adverse effects of interleukin-2.

[0092] Human recombinant interleukin-2 is a well-studied,well-characterized and effective antineoplastic drug with welldocumented, often severe, and sometimes life-threatening or fatal sideeffects. One of the most serious is VLS, which can affect the entirebody and virtually every body system, organ, or tissue.

[0093] According to the “package insert” provided by ChironTherapeutics, IL-2 (PROLEUKIN(®) is a highly purified protein with amolecular weight of approximately 15,300 Daltons. The chemical name isdes-alanyl-1, serine-125 human interleukin-2. IL-2, a lymphokine, isproduced by recombinant DNA technology using a genetically engineered E.coli strain containing an analogue of the human interleukin-2 gene.Genetic engineering techniques were used to modify the human IL-2 gene,and the resulting expression clone encodes a modified humaninterleukin-2. This recombinant form differs from the nativeinterleukin-2 in the follow ways: a) IL-2 is not glycosylated because itis derived from E. coli; b) the molecule has no N-terminal alanine; thecodon for this amino acid was deleted during the genetic engineeringprocedure; c) the molecule has serine substituted for cysteine at aminoacid position 125; this was accomplished by site specific manipulationduring the genetic engineering procedure; and d) the aggregation stateof PROLEUKIN®is likely to be different from that of nativeinterleukin-2.

[0094] In addition, Chiron Therapeutics indicates that certain in vitrostudies were performed to determine the properties of PROLEUKIN® andthat these include: a) enhancement of lymphocyte mitogenesis andstimulation of long-term growth of human interleukin-2 dependent celllines; b) enhancement of lymphocyte cytotoxicity; c) induction of killercell (lymphokine-activated [LAK] and natural [NK] activity; and d)induction of interferon-gamma production. In in vivo studies, IL-2produces multiple immunological effects in murine models in adose-dependent manner. These include: a) activation of cellular immunitywith profound lymphocytosis, eosinophilia, and thrombocytopenia; b) theproduction of other cytokines such as tumor necrosis factor,interleukin-1, and gamma interferon; c) inhibition of tumor growth. Inaddition, as noted previously, interleukin-2 has now been shown tostimulate the production of potentially toxic and inflammatoryleukotriene B₄. Despite the large amount of knowledge concerning theeffects of IL-2, the exact mechanism by which IL-2 mediates itsantitumor (and toxic) effects in humans is unknown.

[0095] A compound useful in this invention is administered to anappropriate subject in need of these compounds by a medically acceptableroute of administration such as orally, parenterally (e.g.,intramuscularly, intravenously, subcutaneously, interperitoneally),transdermally, rectally, by inhalation and the like.

[0096] Unit doses or multiple dose forms are contemplated, each offeringadvantages in certain clinical settings. The unit dose would contain apredetermined quantity of active compound calculated to produce thedesired effect(s) in the setting of IL-2 coadministration. The multipledose form may be particularly useful when multiples of single doses, orfractional doses, are required to achieve the desired ends. Either ofthese dosing forms may have specifications that are dictated by ordirectly dependent upon the unique characteristic of the particularcompound, the particular therapeutic effect to be achieved (e.g., theattenuation of IL-2 toxicity, especially VLS), and any limitationsinherent in the art of preparing the particular compound for treatmentof IL-2 toxicity or VLS in living subjects.

[0097] A unit dose will contain an amount sufficient to mitigate theadverse effects induced by excess IL-2 in a subject and may contain fromabout 5.0 to 1000 mg of compound with the preferred range being 50 to350 mg. The multiple dose form could contain from 0.2 to 4,000 mg withthe preferred range being 100 to 600 mg.

[0098] The compound will preferably be administered orally in a suitableformulation as an ingestible tablet, a buccal tablet, capsule, caplet,elixir, suspension, syrup, trouche, wafer, lozenge, and the like.Generally, the most straightforward formulation is a tablet or capsuleAd(individually or collectively designated as an “oral dosage unit”).Suitable formulations are prepared in accordance with a standardformulating techniques available that match the characteristics of thecompound to the excipients available for formulating an appropriatecomposition. A tablet or capsule will contain about 25 to about 500 mgof a compound of Formula (I), preferably about 50-250 mg, and mostpreferably about 100-200 mg.

[0099] The form may deliver the LA rapidly or may be a sustained-releasepreparation. The LA may be enclosed in a hard or soft capsule, may becompressed into tablets, or may be incorporated with beverages, food orotherwise into the diet. The percentage of the final composition and thepreparations may, of course, be varied and may conveniently rangebetween 1 and 70% of the weight of the final form, e.g., tablet. Theamount of LA in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions according tothe current invention are prepared so that an oral dosage unit formcontains between about 2.5 to about 50 LA by weight (% w) in dosageunits weighing between 50 and 1000 mg.

[0100] The suitable formulation of an oral dosage unit may also contain:a binder, such as gum tragacanth, acacia, corn starch, gelatin;sweetening agents such as lactose or sucrose; disintegrating agents suchas corn starch, alginic acid and the like; a lubricant such as magnesiumstearate; or flavoring such a peppermint, oil of wintergreen or thelike. Various other material may be present as coating or to otherwisemodify the physical form of the oral dosage unit. The oral dosage unitmay be coated with shellac, a sugar or both. Syrup or elixir may containthe LA, sucrose as a sweetening agent, methyl and propylparabens as apreservative, a dye and flavoring. Any material utilized should bepharmaceutically-acceptable and substantially non-toxic. Details of thetypes of excipients useful may be found in the nineteenth edition of“Remington: The Science and Practice of Pharmacy,” Mack PrintingCompany, Easton, Pa. See particularly chapters 91-93 for a fullerdiscussion.

[0101] A compound may be administered parenterally, e.g., intravenously,intramuscularly, intravenously, subcutaneously, or interperitonieally.The carrier or excipient or excipient mixture can be a solvent or adispersive medium containing, for example, various polar or non-polarsolvents, suitable mixtures thereof, or oils. As used herein “carrier”or “excipient” means a pharmaceutically acceptable carrier or excipientand includes any and all solvents, dispersive agents or media,coating(s), antimicrobial agents, iso/hypo/hypertonic agents,absorption-modifying agents, and the like. The use of such substancesand the agents for pharmaceutically active substances is well known inthe art. Except in so far as any conventional media or agent isincompatible with the active ingredient, use in therapeutic compositionsis contemplated. Moreover, other or supplementary active ingredients canalso be incorporated into the final composition. The dosage of theparenteral dosage unit will be 0.1-100% of the oral dosage unit,preferably 10-100%, more preferably 30-100%, and most preferably50-100%.

[0102] Solutions of the LA may be prepared in suitable diluents such aswater, ethanol, glycerol, liquid polyethylene glycol(s), various oils,and/or mixtures thereof, and others known to those skilled in the art.

[0103] The pharmaceutical forms suitable for injectable use includesterile solutions, dispersions, emulsions, and sterile powders. Thefinal form must be stable under conditions of manufacture and storage.Furthermore, the final pharmaceutical form must be protected againstcontamination and must, therefore, be able to inhibit the growth ofmicroorganisms such as bacteria or fungi. A single intravenous orintraperitoneal dose can be administered. Alternatively, a slow longterm infusion or multiple short term daily infusions may be utilized,typically lasting from 1 to 8 days. Alternate day or dosing once everyseveral days may also be utilized.

[0104] Sterile, injectable solutions are prepared by incorporating acompound in the required amount into one or more appropriate solvents towhich other ingredients, listed above or known to those skilled in theart, may be added as required. Sterile injectable solutions are preparedby incorporating the compound in the required amount in the appropriatesolvent with various other ingredients as required. Sterilizingprocedures, such as filtration, then follow. Typically, dispersions aremade by incorporating the compound into a sterile vehicle which alsocontains the dispersion medium and the required other ingredients asindicated above. In the case of a sterile powder, the preferred methodsinclude vacuum drying or freeze drying to which any required ingredientsare added.

[0105] In all cases involving an injectable product the final form, asnoted, must be sterile and must also be able to pass readily through aninjection device such as a hollow needle. The proper viscosity may beachieved and maintained by the proper choice of solvents or excipients.Moreover, the use of molecular or particulate coatings such as lecithin,the proper selection of particle size in dispersions, or the use ofmaterials with surfactant properties may be utilized.

[0106] Prevention or inhibition of growth of microorganisms may beachieved through the addition of one or more antimicrobial agents suchas chlorobutanol, ascorbic acid, parabens, thermerosal, or the like. Itmay also be preferable to include agents that alter the tonicity such assugars or salts.

[0107] Another aspect of this invention is an article of manufacturethat comprises a pharmaceutical composition comprising a leukotriene B₄antagonist, preferably a leukotriene B4 receptor antagonist (asdescribed herein), as a unit dosage in combination with printed labelinginstructions for administering the composition to a mammal undergoingtreatment of a malignancy or viral disease with IL-2, wherein the amountof the composition administered is sufficient to reduce IL-inducedadverse pharmacological effects in the mammal being treated.

[0108] There are several advantages of this invention. It reduces orobviates the need to place patients into intensive care units and ontorespirators in the case of severe pulmonary edema, or to place patientsinto cardiac or coronary care units in the case of severe arrhythmias orcongestive heart failure or onto dialysis protocols in the case of renalcompromise. It reduces intensive nursing care or supportive care or needof ICUs or CCUs. The mitigation of IL-2-related adverse events enablesthe safe administration of higher doses of IL-2 such that the antitumorefficacy of the combined regimen (LA+IL-2) is superior to IL-2 alone. Itreduces diagnostic testing required to monitor patient responses to IL-2and to determine the success of therapeutic interventions required tomitigate IL-2-related adverse events. It reduces diagnostic testingneeded to demonstrate that certain events are caused by IL-2 rather thanby another agent. It reduces the costs associated with diagnosing ortreating IL-2-induced adverse events, particularly those associated withVLS. Another advantage of this invention is that it preserves orenhances the activity of IL-2 (or, at a minimum, reduces the adverseeffects of IL-2 more than any reduction in activity of IL-2). A furtheradvantage is that it permits the administration of higher and moreeffective doses of IL-2 without increasing the risk of adverse effectsfrom IL-2, especially VLS.

[0109] It can be seen from the foregoing discussion that my inventionimproves the benefit to risk ratio and can be said to improve thetherapeutic index. Therapeutic index, in its most general form, relatesthe benefits derived from a particular treatment or therapy to the risksassociated with that same treatment or therapy. Somewhat moremathematically, the therapeutic index may be calculated as the dose ordose level of a drug that provides useful clinical benefit as comparedto the dose or dose level of the same drug that causes adverse events ofsuch severity that the adverse-event causing dose is not tolerated. Theratio of these two doses or dose-levels has been described as the“therapeutic index.” Still a third useful definition is the ratio of thechange in an objective benefit to the change in an objective risk causedby some type of intervention during therapy. The intervention could bethe administration of another drug or drugs or the performance of amedical or surgical procedure, or a combination of these. Those skilledin the art will recognize that other definitions may exist which,nevertheless, connote the fundamental concepts described herein.

[0110] Most simply, a therapeutic index changes if the benefits changebut the risks do not, or the risks change but the benefits do not.However, it is possible that risks and benefits may changesimultaneously, in the same or opposite directions and with similar ordifferent magnitudes. Then the direction and relative magnitude of thechanges become determinant regarding the effect on the therapeuticindex.

[0111] The possible changes in risks and benefits and the effect ontherapeutic index are shown in the table below. TABLE 1 Effect of anIntervention on Benefits, Risks, and Therapeutic Index Change inBenefits Change in Risks Therapeutic Index increase no change increaseincrease decrease increase no change increase decrease decrease nochange decrease decrease increase decrease no change decrease increaseincrease increase increase (less than increase in change in benefits)increase increase decrease (more than increase in change in benefits)decrease decrease decrease (less than decrease in change in benefits)decrease decrease increase (more than decrease in change in benefits)

[0112] Note, however, that in order to determine the effect ontherapeutic index of any intervention that effects either risks orbenefits it is essential to determine the changes in direction andmagnitude of both risks and benefits. No useful statement regarding theimpact of an intervention on therapeutic index can be made withoutknowing simultaneously the effects on risks and benefits.

[0113] All medical judgments regarding the utility of a particulartherapeutic or medical or surgical intervention are made on the basis ofthe therapeutic or interventional impact on the therapeutic index. Thisreliance on therapeutic index may be explicit or implicit but it isinvariant in medical decision-making. I have found that the use of LTB₄antagonist (particularly a LTB₄ receptor antagonist) will improve thetherapeutic ratio of IL-2 treatment by increasing the benefits oftreatment while decreasing the risks.

[0114] The effect of an LTB4 receptor antagonist to decrease the risksassociated from IL-2 exposure may be seen in FIG. 1. As shown, whencompared to control values, oxygen levels in the blood decline when IL-2alone is given but do not decline appreciably when IL-2 isco-administered with an LTB4 receptor blocker. Thus, the risks of IL-2have been reduced.

[0115] The effect of an LTB4 receptor blocker to increase the benefitsassociated with IL-2 exposure may be seen in Table 4 (in Example 5). Asshown, the number of pulmonary metastases are fewer when IL-2 isco-administered with an LTB4 receptor blocker than when IL-2 isadministered alone. Thus, the benefits of IL-2 have been preserved orenhanced.

[0116] Expressed more quantitatively, then, the impact on the risks andbenefits of utilizing IL-2 with or without an LTB4 antagonist may becalculated by comparing the change in the number of metastases (benefit)to the change in blood oxygen (risk) when the treated groups arecompared with the control (untreated) group. The therapeutic index inthis setting is expressed as a ratio of the change in number (N) ofmetastases to change in blood oxygenation (mm Hg) and expressed in unitsof “N/mm Hg.”

[0117] These calculations are shown in the table below: TABLE 2 Changesin Change in Therapeutic Metastases (mean) Oxygen (mean) Index Treatment(N) (mm Hg) (N/mm Hg) IL-2 alone −32 −15 2.1 IL-2 + LTB4 −42  −2 21.0 antagonist

[0118] Thus, it may be observed that the therapeutic index is muchimproved, both qualitatively and quantitatively, when IL-2 is used inconjunction with the LTB₄ antagonist than when IL-2 is utilized alone.This is the first practical demonstration of an improvement intherapeutic index of combination therapy with IL-2.

[0119] A compound of particular value in this invention is(±)7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylicacid. The compound is a potent, highly selective leukotriene B₄ receptorantagonist. A summary of its properties appears below.

In Vitro Pharmacology

[0120] Inhibition of LTB₄ binding to human neutrophils

[0121] IC50=0.3 micromolar

[0122] Inhibition of LTB₄ chemotaxis

[0123] range=0.3-3.0 micromolar

[0124] Inhibition of human neutrophil adhesion to LTB₄-stimulatedumbilical vein endothelial cells

[0125] range=0.3-1.0 micromolar

[0126] Inhibition of LTB₄-induced neutrophil granulation

[0127] range=1-3 micromolar

[0128] Inhibition of LTB₄ synthesis

[0129] IC50=2.1 micromolar

[0130] Inhibition of LTA₄ conversion into LTB₄

[0131] IC50=20 micromolar

In Vivo Pharmacology

[0132] Inhibition of LTB₄ chemotaxis in guinea pigs

[0133] ED50=0.6 mg/kg i.g.

[0134] Inhibition of 12 (R)-HETE in guinea pigs

[0135] ED50=20 mg/kg i.g.

[0136] Inhibition of acetic acid colonic inflammation in rats and guineapig

[0137] ED50=20 mg/kg i.g.

[0138] Inhibition of calcium ionophore dermal inflammation in the guineapig ear

[0139] ED50=0.7 mg/ear

[0140] These in vitro and in vivo data establish the potency andselectivity of the preferred compound and are particularly relevant todiminishing, i.e., mitigating the unwanted effects of IL-2. These dataare also particularly relevant to establishing that leukotriene B₄mediated responses, including VLS, whether induced initially byadministration of IL-2 or by other means, are blunted by the preferredcompound.

[0141] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0142] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

EXAMPLE 1

[0143] This example is a reproduction (without formulae) of Example 1from U.S. Pat. 4,889,871 and sets forth a method for making a preferredcompound useful in this invention, 7-[3 -(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3, 4-dihydro-8-propyl-2H-1-benzopyra-2-carboxylic acid.

[0144] (a) 493 mg of methyl7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylate was added to 25 ml ofacetone containing 276 mg of anhydrous potassium carbonate and 282 mg ofmethyl iodide. The mixture was refluxed for about 24 hours and water wasadded and the mixture was then extracted with ethyl acetate. The extractwas dried, the solvent removed under vacuum, and the residual oil waschromatographed over silica gel with a 40/60 mixture of ethylacetate/hexane to provide pure-methyl ether, methyl7-[3[(4-acetyl-3-methoxy-2-propylphenoxy) propoxy]-3,4-dihydro-8-propy-2H-1 -benzopyran-2-carboxylate.

[0145] (b) The methyl ether (340 mg) was dissolved in methanol (5 ml)containing lithium hydroxide (0.7 ml of a 2N LiOH solution in water).The mixture was stirred at room temperature overnight and the solventremoved in vacuo. The residue was partitioned between ethyl acetate and2N HC1 and the organic layer separated and washed with brine.Evaporation of the volatiles in vacuo afforded crude acid. This materialwas purified by silica gel chromatography using ethylacetate/hexane/acetic acid (40:60:0.5) as eluant. The pure product wasrecrystallized from ethyl acetate/hexane to afford 200 mg of product,7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, m.p.65°-68° C.

[0146] Microanalysis: Found: C 69.22, H 7.53. Theory: C 69.40, H 7.49.

[0147] The NMR (CDCI₃) shows a-OCH₃ at δ3.75.

EXAMPLE 2

[0148] This example explains how compound of Formula (I) is administeredto sheep to mitigate the adverse effect of excess IL-2.

[0149] Preparation of Sheep

[0150] Yearling male or female interbred sheep (N Å 24) and weighingapproximately 20 to 40 kg are prepared with chronic lung lymph fistulaeessentially according to a modification of the technique described byStaub et al (see “Relevant Literature,” supra at 6 and as furtherdescribed within reference 2 herein and other references cited withinreference 2). Through a thoracotomy, the efferent duct of the caudalmediastinal lymph node is cannulated, the distal portion of the lymphnode below the level of the inferior pulmonary ligament is ligated, andthe diaphragm around the lymph node is circumferentially cauterized orotherwise securely closed. All visible systemic lymph tributaries to theproximal portion are cauterized or ligated to minimize extra-pulmonarycontamination of collected lymph. A suitable thermistor-tipped pulmonaryarterial and central venous catheter are introduced through the rightinternal jugular vein. The aorta is then cannulated via the adjacentcarotid artery. After a recovery period of a variable number of days,preferably 4-5, when the animals appear vigorous again, are afebrile,and have a steady flow of blood-free lymph, the experiment is conducted.

[0151] Measurement of Cardiopulmonary Function

[0152] To measure cardiopulmonary function, suitable strain-gaugetransducers are used to measure the following pressures: mean arterialpressure (MAP); mean pulmonary wedge pressure (MPAP, and the pulmonaryarterial wedge pressure (PAWP). The pulmonary microvascular pressure(Pmv) is calculated from the Gaar equation where Pmv=PAWP+0.4 ¥(MPAP-PAWP). Pulse rate is determined from the arterial pressuretracings. Cardiac output is measured serially by thermodilution or dyetechniques using suitable, standard equipment. Arterial blood gases, pH,oxygen saturation, and hemoglobin levels are also measured withsuitable, standard laboratory equipment and by spectrophotometry usingextinction coefficients determined for the species being tested.

[0153] Hematology

[0154] Circulating platelets and leukocytes are counted by means ofstandard laboratory equipment or by phase microscopy while leukocytedifferential counts are determined by counting microscopically on ruledstages of Wright's stained blood smears.

[0155] Biochemical Assays

[0156] Concentrations of leukotriene B₄ and thromboxanes are measuredusing standard radioimmunoassays. Such measurements are or can be madeon lung lymph as well. In addition, lymphatic (LY) and plasma (PL) totalprotein concentrations are determined by using standardspectrophotometric techniques. The LY/PL protein ratio is calculated andmultiplied by lymph flow (QL) to obtain the lymphatic protein clearancerate.

[0157] IL-2

[0158] The recombinant interleukin-2 (PROLEUKIN®) is utilized. The doseof IL-2 can be varied as desired. The preferred range is 1,000 to1,000,000 IU/kg while the most preferred range is 400,000 to 800,000IU/kg.

[0159] Protocol

[0160] Experiments are or can be performed on conscious, instrumentedsheep having free access to food and water. Sheep are allowed to standor recline as desired. Transducers are re-leveled as necessary toaccommodate the sheep stance or posture. Baseline measurements areobtained for variable lengths of time with the preferred period being atleast one hour.

[0161] Certain animals receive IL-2 alone, 400,000 to 800,000 IU/kg,given either as a bolus injection over one to several minutes or as aslower infusion over several minutes to several hours. Other animalsreceive the same IL-2 treatment but also receive, for example, thecompound. The compound may be given by any of the routes identifiedpreviously. For simplicity in the animal laboratory, intravenous dosingis preferred although oral dosing is also acceptable. Effective doses ofthe LA range from 0.1 mg/kg/day to 10 mg/kg/day, while the preferreddose range is 0.1 to 2.0 mg/kg/day and the most preferred dose range is0.5 to 1.0 mg/kg/day. When given intravenously, the LA may be dosedeither as a bolus injection over one to several minutes or as a slowerinfusion over several minutes to several hours. Maintaining continuousbut not necessarily invariant levels of LA during IL-2 exposure ispreferred but is not necessary. LA dosing may begin before, during, orafter the IL-2 administration. It is preferred to begin LA before IL-2administration and is most preferred to begin dosing at least one hourprior to the start of IL-2 administration. The purpose of thisprophylactic regimen is to establish effective blockade of theleukotriene B₄ receptors prior to IL-2-induced increases in plasmalevels of leukotriene B₄.

[0162] Cardiopulmonary function, lymph flows and protein content,hematology and other chemical laboratory tests are assessed during andfor several hours after IL-2 administration begins. In this model it ispreferred to monitor animals as described for a period of 4-6 hours.

[0163] Results

[0164] The in vitro and in vivo results demonstrate the effectiveness ofthe referenced compound in mitigating, reducing or eliminatingleukotriene B₄ induced biological responses. Appropriate analysis of thedata from the ovine experiment described herein extend the beneficialeffects of LA's, LA's preferably, and the referenced LA most preferably,to the setting of preventing IL-2-induced VLS and adverse alterations incardiopulmonary function and laboratory testing.

EXAMPLE 3

[0165] This example explains how a compound of Formula (I) isadministered to rats to mitigate the adverse effect of excess IL-2 inrats.

[0166] Test Material

[0167] A preferred LA compound, (±)7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, (LotNumber 101-8802) was supplied as a white crystalline solid byBioMedicines, Inc. For intraperitoneal (i.p.) injection, the LA wasfreshly dissolved in a mixture of 0.1 M potassium phosphate buffer (pH6.8), ethanol, and propylene glycol (65:20:15 by volume). Theconcentration of the LA solution was adjusted so that the volume foreach injection was 3 ml/kg for all groups.

[0168] Proleukin® for Injection (Chiron Therapeutics) recombinant humaninterleukin-2 (IL-2), is supplied as a lyophilized powder in vialscontaining 22 million international units (MIU). For intravenous (i.v.)injection, Proleukin was reconstituted with Sterile Water for Injection,USP, to provide a solution of 18 million IU/ml. The reconstitutedsolution was further diluted with 5% Dextrose Injection, USP as neededso that the volume for each injection was 0.208 ml/rat. The dose can bevaried as usual. The preferred range for purposes of this example is6-24 MIU/kg while the most preferred range is 10-15 MIU/kg. Any of thepreviously mentioned routes of administration may be used. The preferredroute is i.v. or intraperitoneal (i.p.) while the most preferred, forpurposes of this example, is i.v.

[0169] Animals

[0170] Male Sprague Dawley rats were obtained from Harlan SpragueDawley, Inc., Indianapolis, Indiana. All rats were six to eight weeksold and weighed approximately 250 to 300 g when used in the experiments.Each animal was numbered on the tail with indelible ink. The animalswere housed in groups of up to three per cage and were allowed access tostandard chow and water ad libitum. The study was conducted at an AAALACaccredited facility in compliance with the National Institutes of Health“Guide for the Care and Use of Laboratory Animals,” Publication No.86-23 and the USDA Laboratory Animal Welfare Act, Publication L. 89-544.

[0171] Experimental Protocol

[0172] Six groups of animals were studied (total N=57) in a modificationof the technique of Edwards et al (Edwards MJ, Miller FN, Simms DE etal. Interleukin-2 Acutely Induces Platelet and neutrophil-EndothelialAdherence and Macromolecular Leakage. Cancer Research 52: 1992:3425-31).Animals were weighed on the day of dosing and the appropriate doses ofIL-2 and/or LA were calculated. The animals remained consciousthroughout the experiment until an anesthetic was administeredapproximately 10 minutes before exsanguination. The anesthetic consistedof a mixture of ketamine (45 mg/ml) xylazine (2.5 mg/ml), andacepromazine (0.37 mg/ml) diluted with water and administeredsubcutaneously at a dose of 2.0 ml/kg.

[0173] Group 1 (n=14) consisted of control animals that received bothi.v. and i.p. injections of vehicle. The other five groups received i.v.injections of IL-2 via the tail vein. Groups 2 (n=8), 3 (n =4), and 4(n=10) received IL-2 in doses of 1.5, 6.0, and 12.5 million IU(MIU)/kg,respectively. In addition to 12.5 million IU/kg of IL-2, Groups 5 (n=8)and 6 (n=13) received i.p. injections of the LA. The LA was administeredin two equal, divided doses; the first dose of LA was given 20 minutesbefore administration of IL-2 and the second dose was given 50 minutesafter administration of IL-2. Group 5 was administered 2×10 mg/kg of theLA. Group 6 was administered 2×30 mg/kg of the LA.

[0174] Two hours after the IL-2 injection, the animals wereexsanguinated via the abdominal aorta and the arterial blood wascollected for measurement of PaO2.

[0175] Measurement of Blood Oxygen Levels

[0176] Whole arterial blood was collected into heparinized bloodarterial gas syringes.

[0177] Immediately after collection, the blood sample was analyzed usingPaO2 electrode model DO-166FT. Sample blood was pre-oxygenated and usedas a standard before and after each test sample.

[0178] Analysis of Data

[0179] Data are shown as mean ±SEM. Data were analyzed using theTukey-Kramer test (JMP®, statistical software from SAS, Inc.)Significance was defined as a p-value less than 0.05.

[0180] Results of In vivo Study

[0181] Progressively higher doses of interleukin-2 progressively reducedarterial oxygen levels in the rodent. With a dose of 12.5 MIU IL-2,oxygen levels in the rat declined from a control value of 93.0±2.2 mm Hgto 77.5±2.5 mm Hg (p<0.001). When LA was administered as described thereduction in oxygenation induced by IL-2 was mitigated (p>.0.5 vscontrol and p<0.001 vs IL-2 alone for both LA-treated groups; see FIG.1).

[0182] Discussion

[0183] Interleukin-2 predictably lowers arterial oxygenation in humanswhen administered in a sufficiently high dose. This is an adversepharmacological effect. This reduction in oxygenation is attributable toimpaired ventilatory responses that occur, at least in part, because ofthe VLS induced by IL-2. The current in vitro findings demonstrate thatthe LA utilized in this example blocks leukotriene B₄ receptors andthereby reduces leukotriene B₄ activity. Furthermore, the in vivofindings demonstrate that the administration of an LA prevents theclinically important adverse event of hypoxemia associated with exposureto IL-2 and thereby demonstrating a beneficial effect in preventing theVLS caused by IL-2.

EXAMPLE 4

[0184] This example shows that the specific leukotriene B₄ (LTB₄)antagonist used in Example 3 (referred to here as “BioMed 101”)couldprevent VLS in a rat model of IL-2-induced toxicity.

Materials and Methods

[0185] Animals

[0186] Male Sprague Dawley rats were obtained from Harlan SpragueDawley, Inc., Indianapolis, Indiana. All rats were six to eight weeksold and weighed approximately 250 to 300 g when used in the experiments.The study was conducted at an AAALAC accredited facility in compliancewith the National Institutes of Health “Guide for the Care and Use ofLaboratory Animals,” Publication No. 86-23, and the USDA LaboratoryAnimal Welfare Act, Publication L. 89-544.

[0187] Test Materials

[0188] Proleukin® for Injection (Chiron Therapeutics) recombinant humanIL-2 was purchased from a commercial retail supplier. Proleukin issupplied as a lyophilized powder in vials containing 22 millioninternational units (IU). For intravenous (i.v.) injection, Proleukinwas reconstituted with Sterile Water-for-Injection, USP, to provide asolution of 18 million IU/ml. The reconstituted solution was furtherdiluted with 5% Dextrose Injection USP as needed so that the volume foreach injection was 0.208 ml/animal. Vehicle for IL-2 was prepared in asimilar manner.

[0189] Biomed 101 was supplied as a white crystalline solid byBioMedicines, Inc. (Alameda, Calif.). For purposes of subsequentintraperitoneal injection, Biomed 101 was freshly dissolved in a mixtureof 0.1 M potassium phosphate buffer (pH 6.8), ethanol, and propyleneglycol (65:20:15 by volume). The concentration of the Biomed 101solution was adjusted so that the volume for each injection was 3 ml/kgfor all groups. The Biomed 101 vehicle was prepared in a similar mannerwith adjustments made to equalize the volume of injectate.

[0190] Experimental Protocol

[0191] Six groups of animals were studied (total N=59). Animals wereweighed on the day of dosing and the appropriate doses of IL-2 and/orBiomed 101 were calculated. The animals remained conscious throughoutthe experiment until an anesthetic was administered approximately 10minutes before planned exsanguination. The anesthetic consisted of amixture of ketamine (45 mg/ml), xylazine (2.5 mg/ml), and acepromazine(0.37 mg/ml) diluted with water and administered subcutaneously at adose of 2.0 ml/kg.

[0192] IL-2 or IL-2 vehicle was administered by bolus injection via thetail vein. Biomed 101 or Biomed 101 vehicle was administered byintraperitoneal injection. Biomed 101 or the related vehicle wasadministered in two equal doses, the first dose given 20 minutes beforeadministration of IL-2 and the second dose given 50 minutes afteradministration of IL-2. Two hours after the injection of IL-2 or IL-2vehicle, animals were exsanguinated via the abdominal aorta and thearterial blood was collected for measurement of PaO₂.

[0193] Group 1 (n=13) consisted of control animals that receivedinjections of each vehicle, either i.v. or i.p. as appropriate. Theother five groups received i.v. bolus injections of IL-2 via the tailvein. Groups 2 (n=8), 3 (n=5), and 4 (n=13) received IL-2 in doses of1.5, 6.0, and 12.5 million IU/kg, respectively. In addition to 12.5million IU/kg of IL-2, Groups 5 and 6 also received i.p. injections ofBiomed 101 both prophylactically and following IL-2 administration asnoted above. Group 5 (n−12) received two doses of 30 mg/kg of Biomed 101(total=20 60 mg/kg). Group 6 (n=8) received two doses of 10 mg/kg ofBiomed 101 (total=20 mg/kg).

[0194] The study was conducted in two phases. In the first phase of thestudy, animals were treated with rising doses of IL-2 to determine adose of IL-2 that could significantly lower PaO₂. Dosing was completedin Group 1, Group 2, Group 3 and in five animals in Group 4 before anyanimals received Biomed 101. Subsequently, to further control forpotential variability in response, at least two animals were dosed inGroup 4 (IL-2 12.5 MIU/kg alone) on each day that animals were dosed inGroups 5 and 6 (IL-2 12.5 MIU/kg plus one of the two doses of Biomed101).

[0195] Measurement of Blood Oxygen.

[0196] Whole arterial blood was collected into heparinized syringes.Immediately after collection, the blood sample was analyzed using aLazar Research Laboratories flow-through dissolved-oxygen probe. Thecell temperature was set to 37 degrees centigrade. A syringe pump wasset to 0.5 ml/min to introduce the sample. The cell was zeroed using asolution of heparin and isotonic saline equilibrated with 100% nitrogen.The cell was then calibrated using a solution of heparin and isotonicsaline equilibrated with 20% oxygen. After each PaO₂ determination inwhole blood was made, the cell was washed with heparin ad isotonicsaline. A 20% O₂ standard was introduced before each sample to ensureaccurate and reliable measurements.

[0197] Measurement of Biomed 101 Plasma Levels.

[0198] In animals dosed with Biomed 101, plasma levels of Biomed 101were assayed with a validated, high-pressure liquid chromatographicmethod.

[0199] Statistical analysis.

[0200] In previously published studies, IL-2 administration in the ratresulted in average decreases of PaO₂ from approximately 92 mm Hg to 79mm Hg (with an average standard deviation of approximately 6 mm Hg). Wecalculated that studying five animals per group would give approximatelyan 85% power to detect such a difference at a two-sided p-value of 0.05.Sample sizes of 13 animals per group would be necessary to have an 80%power to detect a difference between 79 mm Hg in the interleukin-onlytreatment group and 86 mm Hg in the interleukin plus Biomed 101treatment group with a two-sided p-value of 0.05 assuming standarddeviations of 6 mm Hg in each group.

[0201] The primary comparison of interest was the difference between theaverage PaO₂s in the interleukin-only treatment group and the group(s)treated with Biomed 101. If the data appeared to be normallydistributed, the comparison was to be made using Student's t-test. Ifinspection of the data indicated that assumptions of the Student'st-test were not applicable, a non-parametric test would be used.

[0202] A p-value of 0.05 was to be considered statistically significant.If the mean PaO₂ in the first Biomed 101-treated group was significantlyhigher than the value in the IL-2 only group, it was then planned todecrease the dose of Biomed 101 in the next group of animals; otherwiseit was planned to increase the dose of Biomed 101 in the next group.

[0203] Because only two primary comparisons of interest were planned, itwas not considered necessary to adjust p-values for multiplecomparisons.

Results

[0204] The average PaO2 in animals receiving only the two vehicles was93.0±2.2 mm Hg (mean±the standard error of the mean). Administration ofProleukin® IL-2 at a dose of 1.5 million IU/kg resulted in no observableacute effect on PaO2. Administration of 6 million IU/kg of IL-2 resultedin decreases in PaO₂ in some of the animals. Evaluation of the dataafter five animals had been dosed with 12.5 MIU/kg suggested that themean PaO₂ decreased significantly and approximately 1.8 mm Hg for eachincrease of 1 MIU/kg in IL-2 administered, as shown in FIG. 1.Therefore, the IL-2 dose of 12.5 MIU/kg was chosen for the IL-2comparison group and the same dose of IL-2 was administered to animalsthat also received Biomed 101.

[0205] Mean and median PaO₂ values in animals dosed with 12.5 MIU/kgalone or with Biomed 101 are summarized in the table below. As noted,12.5 MIU/kg IL-2 alone produced a significant fall in PaO₂. Median PaO₂was significantly higher, however, in animals also dosed with Biomed 101and not significantly different than the value observed in controlanimals.

[0206] The mean plasma level (±the standard error of the mean) of Biomed101 associated with these effects on oxygenation was 6.4 (±0.89) μg/mlin the group of animals dosed with 60 mg/kg Biomed 101 and was 2.0(±0.26) μg/ml in the group of animals dosed with 20 mg/kg Biomed 101.

Discussion

[0207] This example directly evaluated the effect of leukotriene B₄(LTB₄) receptor blockade on the toxicity associated with IL-2administration. Based on the results of this example, blockade of theLTB₄ receptor with Biomed 101 appears to mitigate IL-2 vascular toxicityin the rodent as judged by the absence of hypoxemia after high-doseIL-2. Co-administration of Biomed 101 and IL-2 was shown to prevent themarked reduction in blood oxygen levels caused by administration of IL-2alone.

EXAMPLE 5

[0208] The primary objective of this Example was to determine whetherco-administration of Biomed 101 with IL-2 would affect the anti-tumoractivity of IL-2 in a well-studied murine model of B16 melanoma lungmetastases.

Materials and Methods

[0209] Animals

[0210] Female C57BL/6 mice were obtained from Charles River, Raleigh.

[0211] Test Materials

[0212] Proleukin® for Injection (Chiron Therapeutics) recombinant humanIL-2 was purchased from a commercial retail supplier. Proleukin issupplied as a lyophilized powder in vials containing 22 millioninternational units (IU). For intravenous (i.v.) injection, Proleukinwas reconstituted with Sterile Water-for-Injection, USP, to provide asolution of 18 million IU/ml. The reconstituted solution was furtherdiluted with 5% Dextrose Injection USP as needed so that the volume foreach injection was 0.1 mL/10 g body weight. Vehicle for IL-2 wasprepared in a similar manner.

[0213] For this Example, Biomed 101 was supplied as a white crystallinesolid by BioMedicines, Inc (Alameda, Calif.). For purposes of subsequentintraperitoneal injection, Biomed 101 was freshly dissolved in a mixtureof 0.1 M potassium phosphate buffer (pH 6.5), ethanol, and propyleneglycol (65:20:15 by volume). The concentration of the Biomed 101solution was adjusted so that the volume for each injection was 0.1mL/10 g body weight. The Biomed 101 vehicle was prepared in a similarmanner with adjustments made to equalize the volume of injectate. Theintraperitoneal dose of 25 mg/kg bid for 4 days was shown in apreliminary study to be the highest tolerated dose of Biomed 101 intumored mice treated with IL-2.²²

[0214] The B16/BL6 murine melanoma tumor cells for injection wereobtained from in vitro culture. Mice were inoculated with 3×10⁵ cellsintravenously on Day 0.

[0215] Experimental Protocol

[0216] Three groups of animals received test articles as per protocolsummarized in Table 2 below. TABLE 3 Schedule of Treatments Group #Dosage (N) Test Articles (mg/kg/dose) Route Schedule Group 1 IL-2vehicle iv q6hx2, qdx4, 15 min (20) Biomed 101 ip q6hx2, qdx4, 0 minvehicle Group 2 IL-2  1 mg/kg iv q6hx2, qdx4, 15 min (20) Biomed 101 ipq6hx2, qdx4, 0 min vehicle Group 3 IL-2  1 mg/kg iv q6hx2, qdx4, 15 min(20) Biomed 101 25 mg/kg ip q6hx2, qdx4, 0 min

[0217] Tumor cells were injected on Day 0. IL-2 or IL-2 vehicle wasadministered by bolus injection, two injections give 6 hours apart eachday, Day 1 through Day 4. Biomed 101 or Biomed 101 vehicle wasadministered by intraperitoneal injection. Biomed 101 or vehicle wasadministered 15 minutes prior to each IL-2 injection. An additionalgroup of 15 untreated control mice was used for timing the terminationof the study. At Day 10, three untreated control mice were to beeuthanized each day until tumor burden was determined to be optimal forcounting metastatic foci, at which time the remaining rumored and fivenon-rumored mice were to be euthanized and lungs removed fordetermination of body weights, lung weights and numbers of metastaticfoci.

[0218] Statistical analysis

[0219] Anti-tumor activity was assessed by counts of metastatic foci inthe lungs. Comparison among the three treatment groups (Groups 1, 2 and3) was performed using one-way ANOVA (or Kruskal-Wallis one-way analysisof variance on ranks for data with non-normal distribution) with thethree between-group comparisons performed using adjustment for multiplecomparisons.

Results

[0220] All dosage regimens appeared to be well-tolerated. However, therewas one death in Group 2 (Proleukin alone) on Day 4. The combination ofBiomed 101 and Proleukin (Group 3) resulted in a weight loss of 11% onDay 4. The mice from each treatment group (Groups 1, 2 and 3), thefifteen untreated control mice and five non-tumored untreated mice wereeuthanized on Day 10 and their lungs removed. The lungs were weighed andthe number of lung metastases counted.

[0221] The number of lung metastases was significantly lower in Groups 2and 3 than in the vehicle control Group 1 (one-way ANOVA withStudent-Newman-Keuls method for multiple comparisons). The mean numberof lung metastases was numerically lower in the Biomed 101 treated group(56) than in the group receiving IL-2 alone (66), although thedifference was not statistically significant (Wilcoxon). The effects ofeach treatment are summarized in Table 2 below.

[0222] The lung weights were not normally distributed. One-way analysisof ranks was performed using Kruskal-Wallis test. IL-2 alone did notdecrease the lung weights compared with vehicle control. The combinationof Biomed 101 and IL-2 resulted in significantly lower lung weights thantreatment with IL-2 alone (Dunn's method for multiple comparisons). Theresults are summarized in Table 2 below. TABLE 4 Effect of Treatment onLung Metastases and Lung Group # Number of lung mets Median Lung (N)Test Articles Mean (± SEM) Weight (mg) Group 1 Biomed 101 vehicle 98 174(20) IL-2 vehicle (± 9) Group 2 Biomed 101 vehicle 66* 177 (19) IL-2 (±6) Group 3 Biomed 101 56* 167** (20) IL-2 (± 5)

[0223] The mean number of lung metastases was significantly lower in thegroup of animals treated with IL-2 than in the vehicle control group: 66vs.98. The group of animals treated with Biomed 101 in addition to IL-2had a significantly lower mean number of lung metastases than thevehicle control group (56 vs. 98) and a lower mean number of metastasesthan the group treated with IL-2 alone (56 vs. 66) although thedifference between the two groups receiving IL-2 was not statisticallysignificant. Thus, the coadiminstration of the leukotriene receptorantagonist Biomed 101 fully preserves and may enhance the anti-tumoractivity of IL-2. Taken together, then, the data from Example 4 andExample 5 demonstrate that the combination of Biomed 101 and IL-2 issuperior to IL-2 alone, by improving specifically and uniquely thetherapeutic index of IL-2.

EXAMPLE 6

[0224] This example explains how a compound of Formula (I) isadministered to humans to increase the number of antitumor doses of IL-2that can be administered and well tolerated while preventing theincrease in IL-2-induced adverse effects that are typically associatedwith increasing doses of IL-2.

[0225] Test material

[0226] A preferred LA compound (as defined in Example 3) from drugsubstance lot # BA901 was supplied as 25 mg capsules Batch # 99G1 11 byBioMedicines, Inc. Each hard gelatin capsule contained 25 mg LA plusexcipients including lactose hydrous NF; hydroxypropylmethylcellulose2910, 6 cps USP; sodium lauryl sulfate NF; purified water, USP; andsodium chloride.

[0227] Patients

[0228] Patients meeting the following criteria are eligible fortreatment with the LA and IL-2:

[0229] Men or women age 18 years or older

[0230] Pathologically confirmed renal cell carcinoma

[0231] ECOG performance status 0 or 1 and predicted life expectancy of12 weeks or more

[0232] For women, childbearing potential definitively terminated bysurgery, radiation or menopause or child-bearing potential attenuated byuse of an approved contraceptive method (IUD, oral contraceptive, ordouble-barrier device)

[0233] For women capable of becoming pregnant, negative serum beta-HCGpregnancy test within 7 days prior to initiation of Biomed 101 therapy

[0234] Patients meeting any of the following criteria are ineligible fortreatment with LA:

[0235] History of:

[0236] Significant neurological dysfunction including seizures,uncontrolled central nervous systemic metastases, or clinical signs ofother significant neurological diseases

[0237] Active gastrointestinal bleeding

[0238] Signs of hepatic failure including encephalopathy

[0239] History of moderate or severe coronary artery disease (NYHA Class3 or 4)

[0240] Renal insufficiency (serum creatinine>2.0 mg/dL)

[0241] Aspartate aminotransferase, alanine aminotransferase or serumbilirubin levels more than 2.5 times upper limit of normal

[0242] Hemoglobin<9 g/dL

[0243] A platelet count of less than 100,000 platelets per mm3

[0244] A total white blood cell count of less than 2,000 cells per mm3

[0245] Any concurrent infectious disease requiring antimicrobialtreatment

[0246] Pregnant or lactating females

[0247] Usage of an investigational drug within the thirty (30) daysprior to enrollment; or the planned usage of an investigational drugother than Biomed 101 during the course of the current study.

[0248] Prior treatment with the preferred LA in the setting of thisclinical trial.

[0249] Dosing

[0250] Patients received IL-2 600,000 IU/kg by intravenous bolus threetimes daily during 5 days for a planned total of 14 doses. LA wasadministered orally 1 hour prior to each schedule dose of IL-2 as wellas 8 hours after the final scheduled dose. If dose of IL-2 was withheldbecause of IL-2 induced toxicity, dosing with LA continued. Doses of LAranged from 25 mg three times daily (TID) to 75 mg TID. Plasma levels ofLA were obtained at each dose level.

[0251] Clinical Endpoints

[0252] The principal activity endpoint was the relationship between thetotal number of doses of IL-2 that could be administered and welltolerated by patients and the plasma levels of LA. A second endpoint wasthe response rate in patients of the tumor to the combination therapywith the leukotriene B₄ receptor antagonist and IL-2 as judged bycomplete responses (total disappearance of measurable tumor) or partialresponses (a >50% decrease in measurable tumor volume).

[0253] Analysis of Data

[0254] Data were analyzed by examining the significance of thecorrelation between number of doses of IL-2 and LA plasma level(Spearman's rho).

[0255] Results of Clinical Trial

[0256] The plasma level of LA increased as the total daily dose of LAincreased. As the dose of LA was increased, and the plasma level of LArose, the total number of doses of IL-2 that could be well tolerated bypatients also rose (Spearman's rho=0.47, p<0.03). Similarly, the numberof doses of IL-2 that were required to be withheld because of IL-2toxicity decreased as the plasma level of LA increased. In addition, theobjective response rate in these patients was 27.3% (despite no increasein the incidence of side effects). This objective response rate issignificantly higher than the 8-10% historically noted with I1-2treatment alone. Moreover, this response rate of 27.3% is comparable tothe objective response rate noted in patients treated with thecombination of IL-2 and alpha interferon but for which the[IL-2+interferon] side effect rate is far higher than for IL-2 alone.Thus the combination of IL-2+Biomed 101 is uniquely superior to bothmonotherapy with IL-2 alone or with other combination therapy such asIL-2+alpha interferon.

The subject matter claimed is:
 1. A method of mitigating an adversepharmacological effect of IL-2 when administered to a human as part ofthe treatment of a malignancy or a viral disease, which method comprisesadministering to the human an amount of a leukotriene B₄ (LTB₄)antagonist that is sufficient to mitigate the adverse effect.
 2. Themethod of claim 1, wherein the LTB₄ antagonist is a LTB₄ receptorantagonist.
 3. The method of claim 2, wherein the LTB₄ receptorantagonist mitigates the IL-2-induced adverse effects while preservingor enhancing the antitumor, antiviral, or immunostimulatory effects ofIL-2.
 4. The method of claim 2, wherein the LTB₄ receptor antagonistreduces the IL-2-induced adverse effects more than any reduction in IL-2related antitumor, antiviral, or immunostimulatory effects of IL-2. 5.The method of claim 2, wherein the adverse pharmacological effect isincreased vascular permeability.
 6. The method of claim 5, wherein thesigns and symptoms of the adverse pharmacological effect include edema,systemic hypotension, reduced organ perfusion, or dysfunction of one ormore organs, tissues or cells of the subject body.
 7. The method ofclaim 2, wherein the leukotriene B₄ receptor antagonist is representedby the formula

or a stereoisomer or a pharmaceutically acceptable salt thereof, whereinR¹ represents hydrogen, alkyl having 2 to 6 carbon atoms, alkenyl having2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, or (CH₂)_(n)Rwherein R represents cycloalkyl of 3 to 5 carbon atoms and n is 1 or 2;R2 represents hydrogen, methyl or ethyl; R³ represents alkyl having 1 to5 carbon atoms; W represents (CH₂)_(x) where x is 2 to 7, alkenylenehaving 3 to 7 carbon atoms, alkynylene having 3 to 7 carbon atoms orcyclopentylene; R⁴ represents hydrogen, alkyl having 2 to 5 carbonatoms, alkynyl having 2 to 5 carbon atoms, alkenyl having 2 to 5 carbonatoms, alkanoyl of 2 to 5 carbon atoms or aralkanoyl of 7 to 9 carbonatoms; R⁵ represents hydrogen, alkyl having 1 to 6 carbon atoms, or R⁵represents alkanoyl having 2 to 4 carbon atoms, or (CH₂)_(y)-CO₂R⁸wherein Y is 0 to 4 and R⁸ is hydrogen or having 1 to 6 carbon atoms; R⁶represents hydrogen or together with R⁵ represents a carbon to carbonbond; and A represents-Z-CO₂R⁷ wherein R⁷ represents hydrogen or alkylhaving 1 to 6 carbon atoms, and wherein Z is absent or representsstraight or branched chain alkylene or alkenylene having up to 6 carbonatoms.
 8. The method of claim 7, wherein the leukotriene B₄ receptorantagonist is represented by Formula (I) and R¹ represents alkyl having2 to 4 carbon atoms; R2 represents hydrogen, methyl or ethyl; R³represents alkyl having 1 to 3 carbon atoms; W represents (CH₂), where xis 3 to 5, alkenylene having 3 to 5 carbon atoms, alkynylene having 3 to5 carbon atoms, or cyclopentylene; R⁴ represents alkyl having 2 to 4carbon atoms, acetyl or benzoyl; R⁵ represents hydrogen or alkyl having1 to 4 carbon atoms; R6 represents hydrogen; and A represents-Z-C0₂R⁷,wherein R⁷ represents hydrogen or alkyl having 1 to 4 carbon atoms, andwherein Z is absent or represents alkylene having up to 2 carbon atoms.9. The method of claim 8, wherein the leukotriene B₄ receptor antagonistis represented by Formula (I) and R¹ is n-propyl; R² and R³ each ismethyl; W is (CH₂)_(x), where x is 3,4 or 5; R⁴ is 8-n-propyl; R⁵represents hydrogen or alkyl of 1 to 4 carbon atoms; and Arepresents-Z-CO₂R⁷ wherein R⁷ represents hydrogen or alkyl having 1 to 4carbon atoms and Z is absent or represents alkylene having up to 2carbon atoms.
 10. The method of claim 9, wherein the leukotriene B₄receptor antagonist is represented by Formula (I) and W is (CH₂)₃ R⁵ isH and A is (CH₂)_(p)-COOH where p is 0,1 or
 2. 11. The method of claim10, wherein the leukotriene B₄ antagonist is represented by Formula (I)and A is -CO₂H.
 12. The method of claim 8, wherein R¹ is n-propyl, R² ishydrogen, R³ is methyl, W is (CH₂)₅, R⁴ is 6-acetyl, R⁵ is hydrogen, andA is COOH.
 13. A method of treating a malignancy or a viral disease in asubject, which method comprises administering a therapeuticallyeffective amount of IL-2 in conjunction with a leukotriene B₄ (LTB₄)antagonist in an amount sufficient to reduce IL-2-induced adversepharmacological effects.
 14. The method of claim 13, wherein the LTB₄antagonist is a LTB₄ receptor antagonist.
 15. The method of claim 14,wherein the LTB₄ receptor antagonist reduces the IL-2-induced adverseeffects, while preserving or enhancing the antitumor, antiviral, orimmunostimulatory effects of IL-2.
 16. The method of claim 14, whereinthe LTB₄ receptor antagonist reduces the IL-2-induced adverse effectsmore than any reduction in IL-2 related antitumor, antiviral, orimmunostimulatory effects of IL-2.
 17. The method of claim 14, whereinthe adverse pharmacological effect is increased vascular permeability.18. The method of claim 17, wherein the signs and symptoms of theadverse pharmacological effect include one or more of edema, systemichypotension, reduced organ perfusion, or dysfunction of one or moreorgans, tissues or cells of the subject's body.
 19. The method of claim14, wherein the leukotriene B₄ receptor antagonist is represented by theformula

or a stereoisomer or a pharmaceutically acceptable salt thereof whereinR¹ represents alkyl having 2 to 6 carbon atoms, alkenyl having 2 to 6carbon atoms, alkynyl having 2 to 6 carbon atoms, or (CH₂)_(n)R whereinR represents cycloalkyl of 3 to 5 carbon atoms and n is 1 or 2; R²represents hydrogen, methyl or ethyl; R³ represents alkyl having 1 to 5carbon atoms; W represents (CH₂)_(x) where x is 2 to 7, alkenylenehaving 3 to 7 carbon atoms, alkynylene having 3 to 7, or cyclopentylene;R⁴ represents hydrogen, alkyl having 2 to 5 carbon atoms, alkenyl having2 to 5 carbon atoms, alkenyl having 2 to 5 carbon atoms, alkanoyl of 2to 5 carbon atoms, or aralkanoyl of 7-9 carbon atoms; R⁵ representshydrogen, alkyl having 1 to 6 carbon atoms, or R⁵ represents alkanoylhaving 2 to 4 carbon atoms, or (CH₂)_(y)-CO²R⁸ wherein y is 0 to 4 andR⁸ is hydrogen or alkyl having 1 to 6 carbon atoms; R⁶ representshydrogen or together with R ⁵represents a carbon to carbon bond; and Arepresents-Z-CO₂R⁷ wherein R⁷ represents hydrogen or alkyl having 1 to 6carbon atoms, and wherein Z is absent or represents straight or branchedchain alkylene or alkenylene having up to 6 carbon atoms.
 20. The methodof claim 19, wherein the leukotriene B₄ receptor antagonist isrepresented by Formula (I) and R¹ represents alkyl having 2 to 4 carbonatoms; R² represents hydrogen, methyl or ethyl; R³ represents alkylhaving 1 to 3 carbon atoms; W represents (CH₂)_(x) where x is 3 to 5,alkenylene having 3 to 5 carbon atoms, alkynylene having 3 to 5 carbonatoms, or cyclopentylene; R⁴ represents alkyl having 2 to 4 carbonatoms, acetyl or benzoyl; R⁵ represents hydrogen or alkyl having I to 4carbon atoms; R⁶ represents hydrogen; and A represents-Z-CO₂R⁷, whereinR⁷ represents hydrogen or alkyl having 1 to 4 carbon atoms, and whereinZ is absent or represents alkylene having up to 2 carbon atoms.
 21. Themethod of claim 20, wherein the leukotriene B₄ receptor antagonist isrepresented by Formula (I) and R¹ is n-propyl; R² and R³ each is methyl;W is (CH₂)_(x), where x is 3,4 or 5; R₄ is 8-n-propyl; R₅ representshydrogen or alkyl of 1 to 4 carbon atoms; and A represents -Z-CO₂R⁷wherein R⁷ represents hydrogen or alkyl having 1 to 4 carbon atoms and Zis absent or represents alkylene having up to 2 carbon atoms.
 22. Themethod of claim 21, wherein the leukotriene B₄ receptor antagonist isrepresented by Formula (I) and W is (CH₂)₃, R⁵ is H and A is (CH₂)p -COOH where p is 0,1 or
 2. 23. The method of claim 22, wherein theleukotriene B₄ antagonist is represented by Formula (I) and A is -COOH.24. The method of claim 20, wherein R¹ s n-propyl, R² is hydrogen, R³ ismethyl, W is (CH₂)₅, R⁴ is 6-acetyl, R⁵ is hydrogen, and A is COOH. 25.The method of claim 14, wherein the IL-2 and the leukotriene B₄ receptorantagonist are administered in combination as a unit dosage.
 26. Themethod of claim 14, wherein the IL-2 and the leukotriene B₄ receptorantagonist are administered individually.
 27. The method of claim 26,wherein the leukotriene B₄ receptor antagonist is administered orallybefore, during or after the IL-2 is administered.
 28. An article ofmanufacture that comprises a pharmaceutical composition comprising aleukotriene B₄ (LTB₄) antagonist as a unit or multiple dosage incombination with printed labeling instructions for administering thedosage to a subject undergoing treatment of a malignancy or viraldisease with IL-2, wherein the amount of the composition administered issufficient to mitigate IL-induced adverse pharmacological effects in themammal being treated.
 29. The article of manufacture of claim 28,wherein the LTB₄ antagonist is a LTB₄ receptor antagonist.
 30. Thearticle of manufacture of claim 29, wherein the amount of the LTB₄receptor antagonist to be administered mitigates the IL-2-inducedadverse effects while preserving or enhancing the antitumor, antiviral,or immunostimulatory effects of IL-2.
 31. The method of claim 29,wherein the amount of the LTB₄ receptor antagonist reduces theIL-2-induced adverse effects more than any reduction in IL-2 relatedantitumor, antiviral, or immunostimulatory effects of IL-2.
 32. Thearticle of manufacture of claim 29, wherein the adverse pharmacologicaleffect is increased vascular permeability.
 33. The article ofmanufacture of claim 32, wherein the signs and symptoms of the adversepharmacological effect include one or more of edema, systemichypotension, reduced organ perfusion, or dysfunction of one or moreorgans of the mammal's body.
 34. The method of claim 29, wherein theleukotriene B₄ receptor antagonist is represented by the formula

or a stereoisomer or a pharmaceutically acceptable salt thereof whereinR¹ represents alkyl having 2 to 6 carbon atoms, alkenyl having 2 to 6carbon atoms, alkynyl having 2 to 6 carbon atoms, or (CH₂)_(n)R whereinR represents cycloalkyl of 3 to 5 carbon atoms and n is 1 or 2; R²represents hydrogen, methyl or ethyl; R³ represents alkyl having 1 to 5carbon atoms; W represents (CH₂)_(x) where x is 2 to 7, alkenylenehaving 3 to 7 carbon atoms, alkynylene having 3 to 7 carbon atoms, orcyclopentyl; R⁴ represents hydrogen, alkyl having 2 to 5 carbon atoms,alkenyl having 2 to 5 carbon atoms, alkenyl having 2 to 5 carbon atoms,alkanoyl of 2 to 5 carbon atoms, or aralkanoyl of 7 to 9 carbon atoms;R⁵ represents hydrogen, alkyl having 1 to 6 carbon atoms, or R⁵represents alkanoyl having 2 to 4 carbon atoms, or (CH₂)_(y)-CO₂R⁸wherein Y is 0 to 4 and R⁸ is hydrogen or alkyl having 1 to 6 carbonatoms; R⁶ represents hydrogen or, together with R⁵, represents a carbonto carbon bond; and A represents-Z-CO₂R⁷ wherein R⁷ represents hydrogenor alkyl having 1 to 6 carbon atoms, and wherein Z is absent orrepresents straight or branched chain alkylene or alkenylene having upto 6 carbon atoms.
 35. The article of manufacture of claim 34, whereinthe leukotriene B₄ receptor antagonist is represented by Formula (I) andR¹ represents alkyl having 2 to 4 carbon atoms; R² represents hydrogen,methyl or ethyl; R³ represents alkyl having 1 to 3 carbon atoms; Wrepresents (CH₂)_(x) where x is 3 to 5, alkenylene having 3 to 5 carbonatoms, alkynylene having 3 to 5 carbon atoms, or cyclopentylene; R⁴represents alkyl having 2 to 4 carbon atoms, acetyl or benzoyl; R⁵represents hydrogen, alkyl having 1 to 4 carbon atoms; R⁶ representshydrogen; and A represents-Z-CO₂R⁷, wherein R⁷ represents hydrogen oralkyl having 1 to 4 carbon atoms, and wherein Z is absent or representsalkylene having up to 2 carbon atoms.
 36. The article of manufacture ofclaim 35, wherein the leukotriene B₄ receptor antagonist is representedby Formula (I) and R¹ is n-propyl; R² and R³ each is methyl; W is(CH2)x, where x is 3,4 or 5; R⁴ is 8-n-propyl; R⁵ represents hydrogen oralkyl of 1 to 4 carbon atoms; and A represents-Z-CO₂R⁷ wherein R⁷represents hydrogen or alkyl having 1 to 4 carbon atoms and Z is absentor represents alkylene having up to 2 carbon atoms.
 37. The article ofmanufacture of claim 36, wherein the leukotriene B4 receptor antagonistis represented by Formula (I) and W is (CH₂)₃, R⁵ is H and A is(CH₂)_(p)-COOH where p is 0,1 or
 2. 38. The article of manufacture ofclaim 37, wherein the leukotriene B₄ antagonist is represented byFormula (I) and A is-COOH.
 39. The article of manufacture of claim 34,wherein R¹ is n-propyl, R² is hydrogen, R³ is methyl, W is (CH₂)₅, R⁴ is6-acetyl, R⁵ is hydrogen, and A is COOH.
 40. The article of manufactureof claim 29, wherein the composition comprises IL-2 and the leukotrieneB₄ receptor antagonist in combination as a unit dosage.
 41. The articleof manufacture of claim 29, wherein the labeling instructions indicatethat the IL-2 and the leukotriene B₄ receptor antagonist areadministered individually.
 42. The article of manufacture of claim 29,wherein the labeling instructions indicate that the leukotriene B₄receptor antagonist is administered orally before, during or after theIL-2 is administered.
 43. A process for preparing a pharmaceuticalcomposition useful for mitigating an adverse pharmacological effect ofIL-2 in a subject, which process comprises (a) combining a leukotrieneB₄ (LTB₄) antagonist with a pharmaceutically acceptable excipient, (b)packaging the resulting combination with printed labeling instructionsfor administering the combination to a subject undergoing treatment of amalignancy or a viral disease with IL-2, wherein the amount of thecombination indicated to be administered is sufficient to mitigateIL-induced adverse pharmacological effects in the subject being treated.44. The process of claim 43, wherein the LTB₄ antagonist is a LTB₄receptor antagonist.
 45. The process of claim 44, wherein the amount ofthe LTB₄ receptor antagonist to be administered mitigates theIL-2-induced adverse effects while preserving or enhancing theantitumor, antiviral, or immunostimulatory effects of IL-2.
 46. Theprocess of claim 44, wherein the amount of the LTB₄ receptor antagonistto be administered reduces the IL-2-induced adverse effects more thanany reduction in IL-2 related antitumor, antiviral, or immunostimulatoryeffects of IL-2.
 47. The method of claim 44, wherein the leukotriene B₄receptor antagonist is represented by the formula

or a stereoisomer or a pharmaceutically acceptable salt thereof, whereinR¹ represents hydrogen, alkyl having 2 to 6 carbon atoms, alkenyl having2 to 6 carbon 1-5 atoms, alkynyl having 2 to 6 carbon atoms, or(CH₂)_(n)R wherein R represents cycloalkyl of 3 to 5 carbon atoms and nis 1 or 2; R2 represents hydrogen, methyl or ethyl; R³ represents alkylhaving 1 to 5 carbon atoms; W represents (CH₂)_(x) where x is 2 to 7,alkenylene having 3 to 7 carbon atoms, alkynylene having 3 to 7 carbonatoms or cyclopentylene; R⁴ represents hydrogen, alkyl having 2 to 5carbon atoms, alkynyl having 2 to 5 carbon atoms, alkenyl having 2 to 5carbon atoms, alkanoyl of 2 to 5 carbon atoms or aralkanoyl of 7-9carbon atoms; R⁵ represents hydrogen, alkyl having 1 to 6 carbon atoms,or R⁵ represents alkanoyl having 2 to 4 carbon atoms, or (CH₂)_(y)-CO₂R⁸wherein Y is 0 to 4 and R⁸ is hydrogen or alkyl having 1 to 6 carbonatoms; R⁶ represents hydrogen or, together with R⁵, represents a carbonto carbon bond; and A represents-Z-C0₂R⁷ wherein R⁷ represents hydrogenor alkyl having 1 to 6 carbon atoms, and wherein Z is absent orrepresents straight or branched chain alkylene or alkenylene having upto 6 carbon atoms.
 48. The process of claim 47, wherein the leukotrieneB₄ receptor antagonist is represented by Formula (I) and R¹ representsalkyl having 2 to 4 carbon atoms; R² represents hydrogen, methyl orethyl; R³ represents alkyl having 1 to 3 carbon atoms; W represents(CH₂)_(x) where x is 3 to 5, alkenylene having 3 to 5 carbon atoms,alkynylene having 3 to 5 carbon atoms, or cyclopentylene; R⁴ representsalkyl having 2 to 4 carbon atoms, acetyl or benzoyl; R⁵ representshydrogen or alkyl having I to 4 carbon atoms; R⁶ represents hydrogen;and A represents-Z-CO₂R⁷, wherein R⁷ represents hydrogen or alkyl having1 to 4 carbon atoms, and wherein Z is absent or represents alkylenehaving up to 2 carbon atoms.
 49. The process of claim 48, wherein theleukotriene B₄ receptor antagonist is represented by Formula (I) and R¹is n-propyl; R² and R³ each is methyl; W is (CH₂)_(x), where x is 3,4 or5; R⁴ is 8-n-propyl; R⁵ represents hydrogen or alkyl of I to 4 carbonatoms; and A represents-Z-CO₂R⁷ wherein R⁷ represents hydrogen or alkylhaving 1 to 4 carbon atoms and Z is absent or represents alkylene havingup to 2 carbon atoms.
 50. The process of claim 49, wherein theleukotriene B₄ receptor antagonist is represented by Formula (I) and Wis (CH₂)₃, R⁵ is H and A is (CH₂)_(p)-COOH where p is 0,1 or
 2. 51. Theprocess of claim 50, wherein the leukotriene B₄ antagonist isrepresented by Formula (I) and A is-CO₂H.
 52. The process of claim 48,wherein R¹ is n-propyl, R² is hydrogen, R³ is methyl, W is (CH₂)₅, R⁴ is6-acetyl, R⁵ is hydrogen, and A is COOH.